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. 2020 Nov 19;15(6):1333–1346. doi: 10.1016/j.stemcr.2020.10.010

Unique Functional Properties of Mature Adult-Born Neurons in the Mouse Olfactory Bulb

Natalie Fomin-Thunemann 1, Yury Kovalchuk 1, Stefan Fink 1, Astrid Alsema 1, Nima Mojtahedi 1, Elizabeta Zirdum 1, Olga Garaschuk 1,∗
PMCID: PMC7724478  PMID: 33217326

Summary

The rodent olfactory bulb (OB) is continuously supplied with adult-born cells maturing into GABAergic neurons. Using in vivo ratiometric Ca2+ imaging to readout ongoing and sensory-driven activity, we asked whether mature adult-born cells (mABCs) in the glomerular layer of the bulb become functionally identical to resident GABAergic (ResGABA) neurons. In awake head-restrained mice the two cell populations differed significantly in terms of ongoing spontaneous activity, with 24% of mABCs contributing to a strongly active cell cluster, absent among ResGABA cells. Odor-evoked responses of mABCs were sparse, less reliable, and had smaller amplitudes compared with ResGABA cells. The opposite was seen under anesthesia, with response reliability increasing and response size of mABCs becoming larger than that of ResGABA cells. Furthermore, ongoing activity of mABCs showed increased sensitivity to ketamine/xylazine and was selectively blocked by the antagonist of serotonin receptors methysergide. These functional features of mABCs clearly distinguish them from other OB interneurons.

Keywords: awake mice, in vivo calcium imaging, adult-born cells, serotonin, 5-hydroxytryptamin, noradrenaline, acetylcholine, olfactory bulb, GABAergic cells, odor responses

Highlights

  • •

    In awake mice mature adult-born neurons differ from resident GABAergic cells

  • •

    24% of neurons belong to a strongly active cell cluster, absent among resident cells

  • •

    Serotonergic inputs target adult-born but not resident GABAergic cells

In this article, Garaschuk and colleagues use in vivo ratiometric calcium imaging to show that mature adult-born cells in the glomerular layer of the mouse olfactory bulb differ from resident GABAergic neurons in terms of spontaneous ongoing activity, odor-evoked responses, sensitivity to ketamine/xylazine anesthesia, and unique serotonergic inputs.

Introduction

The rodent olfactory bulb (OB) is continuously supplied with adult-born cells (ABCs). These cells are implicated in odor discrimination; facilitation of the task-dependent pattern separation; short- and long-term olfactory memory; perceptual, associative olfactory, and fear learning (Alonso et al., 2006; Li et al., 2018; Moreno et al., 2009; Pan et al., 2012; Sultan et al., 2010; Valley et al., 2009). They also contribute to innate olfactory behaviors, such as mating, pregnancy, and offspring recognition (Feierstein et al., 2010; Mak et al., 2007; Sakamoto et al., 2011).

Born in the subventricular zone (SVZ), ABCs migrate via the rostral migratory stream (RMS) toward the OB (Lledo et al., 2008). Ninety percent of these cells become inhibitory granule cells (GCs), while 5%–10% become GABAergic periglomerular (PGCs) or short axon cells (SACs) (Lledo et al., 2008), collectively referred to as juxtaglomerular cells (JGCs). Not all cells survive for longer time periods (Petreanu and Alvarez-Buylla, 2002). In addition to endogenous and sensory-driven activity (Lin et al., 2010; Petreanu and Alvarez-Buylla, 2002; Yamaguchi and Mori, 2005), centrifugal inputs arising from the olfactory cortex, basal forebrain, dorsal raphe nucleus, and locus coeruleus were shown to promote survival of ABCs (Bauer et al., 2003; Deshpande et al., 2013; Kaneko et al., 2006; Mechawar et al., 2004; Siopi et al., 2016; Whitman and Greer, 2007).

During the pre-integration phase, i.e. after arrival to the glomerular layer (GL) but before the integration therein (Liang et al., 2016), functional properties of adult-born JGCs are well known to differ from those of ResGABA cells. These differences include (1) expression pattern of glutamatergic receptors, with ABCs having lower AMPA to NMDA ratios; (2) lower levels of spontaneous activity; (3) higher odor responsiveness; (4) lower response selectivity; and (5) higher structural plasticity (Belluzzi et al., 2003; Grubb et al., 2008; Kovalchuk et al., 2015; Livneh et al., 2009, 2014; Maslyukov et al., 2018; Mizrahi, 2007; Wallace et al., 2017). Immature ABCs are particularly sensitive to environmental influences. Thus, 2–3 weeks old adult-born GCs were more responsive to novel odors than resident GCs but became less responsive over time (Magavi et al., 2005). Consistently, 2–4 weeks old adult-born PGCs showed larger Na+ conductance, had higher odor-evoked action potential (AP) firing rates, and responded more broadly to different odorants compared with 8–9 weeks old or resident cells (Belluzzi et al., 2003; Livneh et al., 2014).

As ABCs mature, their AMPA/NMDA ratio increases (Grubb et al., 2008). From 7 to 8 weeks onward the spontaneous and odor-evoked activity as well as odor-selectivity of ABCs becomes more similar to that of resident cells (Kovalchuk et al., 2015; Livneh et al., 2014; Wallace et al., 2017). Dendritic dynamics decreases and stabilizes at age 6–12 weeks (Livneh et al., 2009; Livneh and Mizrahi, 2011). Therefore, ABCs older than 8 weeks are considered mature. However, the key question remains: Do mature ABCs (mABCs) become functionally identical to ResGABA cells, or do they still preserve unique features, thus increasing the substrate for plasticity and functional diversity of interneurons in the adult OB?

By using in vivo two-photon Ca2+ imaging in awake, head-restrained mice, we compared the functional properties of mABCs and ResGABA cells in the GL of the bulb with respect to their spontaneous and odor-evoked activity. As anesthesia modifies functional properties of interneurons (Kato et al., 2012; Wachowiak et al., 2013), we used it as a tool to probe for those functional differences, which might not be evident under awake conditions. Finally, we tested whether the centrifugal projections target mABCs and ResGABA cells differently.

Results

Ongoing Neuronal Activity of mABCs in Awake State

To monitor the level of spontaneous ongoing activity in mABCs of wild-type (WT) mice, we infected them with lentiviruses expressing the ratiometric Förster resonance energy transfer-based Ca2+ indicator Twitch-2B (Thestrup et al., 2014) (Figure 1A) and calculated a background-corrected ratio of the acceptor to donor fluorophore fluorescence: cpVenusCD/mCerulean3 (hereafter designated as Twitch-2B ratio; Experimental Procedures). Changes in the Twitch-2B ratio correlate with changes in the intracellular free Ca2+ concentration ([Ca2+]i) (Palmer and Tsien, 2006), with higher ratios corresponding to higher levels of [Ca2+]i. In awake mice, the mean basal Twitch-2B ratios, measured in the absence of any exogenous stimuli, ranged from 1.44 to 8.44 (Figures 1B and 1C). As expected (Maslyukov et al., 2018), application of the voltage-gated Na+ channel blocker tetrodotoxin (TTX) through a slit in the cranial window (Note S1) caused a profound decrease in basal Twitch-2B ratios (Figure 1D). Under TTX, basal ratios of the majority of cells dropped below 2. Thus, in further analyses cells with basal Twitch-2B ratios <2 were considered “non-spiking.” Only 18.2% of mABCs were non-spiking in the awake state.

Figure 1.

Figure 1

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Basal Twitch-2B Ratios of mABCs in Awake Mice

(A) Maximum intensity projection of a 3D stack (90–100 μm below the dura, step 2 μm) showing mABCs (131 days post-injection [DPI]) expressing Twitch-2B. Here and in Figure 2A the image is an overlay of the mCerulean3 (green) and cpVenusCD (red) channels.

(B) Ratio traces recorded in cells marked with respective numbers in (A).

(C) Distribution of mean mABC Twitch-2B ratios under control conditions (n = 214 cells, 11 mice).

(D) Distribution of mean mABC Twitch-2B ratios under 5 μM tetrodotoxin (TTX) (n = 37 cells, 2 mice). Note that 94.59% of cells had Twitch-2B ratios <2.0 (vertical broken line). Inset: an overlay image of an mABC before (left, ratio 4.0) and during (right, ratio 1.6) TTX application.

(E) Scatterplot showing the ratios of cells recorded on day 0 (53–71 DPI) plotted against the corresponding ratios recorded 3 or 6 days later (n = 35 cells, 5 mice). Diagonal broken line: y = x.

(F) Boxplot shows the median (per mouse) normalized Twitch-2B ratios (day 3/day 0 and day 6/day 0). The ratios were not different from one (p = 0.31 and 0.19, respectively, Wilcoxon signed rank test, 5 mice).

To accommodate slight ratio changes caused by the background subtraction and to avoid false positive results, we introduced a safety margin between 2 and 2.4. Only cells with mean basal ratios >2.4 were considered “spiking” (see also Figure S1). Cells with ratios 2–2.4 were considered “uncertain.” Using this categorization, 63.1% of mABCs were considered spiking in the awake state. To test the stability of basal ratios of individual cells over time, the latter were measured on the first recording day and re-examined 3 and 6 days later. Some cells showed higher or lower ratios at later time points (Figure 1E), but on a population level we observed a striking stability of mean basal Twitch-2B ratios of mABCs during a week time span (Figure 1F).

Differences in Basal Twitch-2B Ratio between mABCs and ResGABA Cells

As ABCs mature to become GABAergic interneurons, we compared basal Twitch-2B ratios of mABCs and mature ResGABA cells. ResGABA cells were stained by injection of an adeno-associated virus encoding a Cre-inducible version of Twitch-2B into Viaat-Cre mice. Post-hoc immunocytochemistry (Supplemental Experimental Procedures) revealed that these cells comprised 23.61% ± 7.92% of tyrosine hydroxylase-positive, 38.46% ± 16.74% of calretinin-positive, and 19.78% ± 5.63% of calbindin-D-28k-positive cells, similar to the composition of the mABC population analyzed in our previous study (Figure S2) (Kovalchuk et al., 2015).

In awake mice, mean basal ratios of ResGABA cells ranged from 1.27 to 6.05 (Figure 2B). Noteworthy, the cumulative distributions of basal ratios were significantly different between mABCs and ResGABA cells (Figure 2C). We also observed a higher fraction of spiking and a lower fraction of non-spiking cells among mABCs (Figure 2D), but these differences did not reach the level of statistical significance (p = 0.08, chi-square test). According to Bayesian information criterion the distribution of basal Twitch-2B ratios of ResGABA cells was best fitted with two Gaussians, whereas that of mABCs was best fitted with three Gaussians (Figure 2E). The first two Gaussians (mean ± SD: 1.98 ± 0.07 and 3.2 ± 0.75 for ResGABA cells, and 1.96 ± 0.06 and 2.97 ± 0.39 for mABCs, respectively) were similar for both cell populations. However, the fit of the mABCs dataset contained a third component (arrow in Figure 2E; mean ± SD: 5.56 ± 1.5), which was absent in the ResGABA dataset.

Figure 2.

Figure 2

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Comparison of ResGABA and mABC Basal Twitch-2B Ratios in Awake Mice

(A) Mean intensity projection image (250 frames) of ResGABA cells (180 DPI). An arrowhead points to a spiking cell (ratio 4.69) and an arrow to a non-spiking cell (ratio 1.6).

(B) Distribution of Twitch-2B ratios in 382 ResGABA cells (n = 5 mice). Here and below vertical broken lines delineate borders between non-spiking and spiking cell populations.

(C) Cumulative probability histograms of Twitch-2B ratios in mABCs and ResGABA cells (∗p = 2.5 × 10−3, two-sample Kolmogorov-Smirnov test, n = 214 and 382 cells, respectively).

(D) Pie charts showing the fractions of spiking, non-spiking, and uncertain cells among mABCs (top) and ResGABA cells (bottom).

(E) Probability density functions are Gaussian fits of distributions shown in Figures 1C and 2B. The area under the third Gaussian (red arrow) equals 24.1% of the total area under the function.

To relate the measured basal Twitch-2B ratios to cell AP firing frequency, we recorded Twitch-2B ratios of ResGABA cells simultaneously with the underlying APs in in situ calibration experiments (Figure S1). By injecting steady-state currents, we varied median AP frequency from 2.5 to 57 Hz and corresponding Twitch-2B ratios from 2.3 to 7.6. Fitting the data with the Hill equation returned the following parameters: Rmin = 1.8, Rmax = 8.5, half maximal effective concentration = 13.94, and Hill coefficient = 1.47, which are in a good agreement with in vivo data obtained in this and previous studies (Maslyukov et al., 2018; Thestrup et al., 2014).

Differences in Odor-Response Properties between mABCs and ResGABA Cells

To compare odor-evoked Ca2+ signals of mABCs and ResGABA cells in awake mice, we used ethyl tiglate (ETI), known to activate numerous glomeruli in the dorsal OB (Rokni et al., 2014). ETI evoked reproducible Ca2+ transients both in mABCs and ResGABA cells (Figures 3A and 3B). Cells responding at least once in eight trials were termed “responding,” the ones responding in at least five out of eight trials were termed “reliably responding.”

Figure 3.

Figure 3

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Odor-Evoked Responses of mABC and ResGABA Cells in Awake Mice

(A and B) Sample Ca2+ transients evoked by ethyl tiglate (ETI) (1.7% of saturated vapor) in an mABC (A) and a ResGABA cell (B).

(C) Pie charts showing the fractions of reliably responding, non-reliably responding, and non-responding cells among mABCs (top) and ResGABA cells (bottom; p < 10−4, chi-square test; n = 116 mABCs, 8 mice versus n = 222 ResGABA cells, 5 mice).

(D–F) Cumulative probability histograms showing the distributions of the CV (D) (p = 0.39, here and below Kolmogorov-Smirnov test, n = 51 mABCs, 8 mice versus n = 174 ResGABA cells, 5 mice), mean amplitude (E) (∗p = 4 × 10−3), and the maximal odor-evoked Twitch-2B ratios (F) (p = 0.19).

(G) Relationships between the basal and the maximal odor-evoked Twitch-2B ratios (Spearman's rank correlation coefficient r = 0.62, n = 51 mABCs; r = 0.58, n = 174 ResGABA cells, p < 10−4 for both comparisons).

The fractions of reliably responding, non-reliably responding, and non-responding cells differed significantly between mABCs and ResGABA cells, with ResGABA cells having almost twice as many reliably responding cells as mABCs, and mABCs having a three times larger fraction of non-responding cells (Figure 3C). For reliably responding cells, the spread in response amplitude was estimated using the coefficient of variation (CV) (Experimental Procedures). The CVs (Figure 3D) and the odor-evoked maximal ratios (Figure 3F) were similar for mABCs and ResGABA cells. However, the response amplitude of reliably responding mABCs was significantly smaller than that of reliably responding ResGABA cells (Figure 3E). We also observed a significant correlation between the basal and the odor-evoked maximal ratios in both cell groups (Figure 3G).

Effect of Anesthesia on Basal Ca2+ Levels of mABCs and ResGABA Cells

Anesthesia has a profound effect on the activity of GABAergic interneurons (Kato et al., 2012; Wachowiak et al., 2013). We used this fact to test for further differences between mABCs and ResGABA cells. In separate experiments we applied three commonly used anesthesia regimens (medetomidine/midazolam/fentanyl (MMF), ketamine/xylazine (K/X), and isoflurane) and compared the Twitch-2B ratios of the same cells under two conditions: wakefulness and subsequent anesthesia. All anesthesia regimens strongly reduced basal Twitch-2B ratios in mABCs (Figures S3A–S3C). Accordingly, the fractions of spiking and non-spiking cells were almost reversed under the anesthesia (insets in Figures S3A–S3C). To compare the effects of the three regimens, we related the cell's change in the basal ratio (from awake to anesthesia) to the maximal possible change and termed this value “effect size” (Experimental Procedures). The median (per mouse) effect sizes for all anesthesia regimens were large and significantly different from zero (MMF: 56.26%; K/X: 73.73%; isoflurane: 70.86%, p < 0.01 for all comparisons, n = 8 mice, one-tailed Wilcoxon signed rank test) with no difference between the three effect sizes (Figure S3D).

In ResGABA cells, anesthesia also strongly reduced the basal Twitch-2B ratios, with cells shifting significantly from spiking to non-spiking state (Figures S4A–S4C). The median (per mouse) effect sizes significantly differed from zero (MMF: 39.73%; K/X: 46.60%; isoflurane: 66.01%, p < 0.05, n = 5 mice, one-tailed Wilcoxon signed rank test). For ResGABA cells, however, the effect sizes of the three regimens were significantly different (Figure S4D).

Next, we compared the effect of anesthesia on mABCs versus ResGABA cells. Under all three regimens basal Twitch-2B ratios of mABCs were lower than those of ResGABA cells (Figures 4A–4C), but the difference reached the level of statistical significance only for K/X anesthesia (Figure 4B). Moreover, the fractions of spiking, non-spiking, and uncertain cells differed between mABCs and ResGABA cells under K/X (inset in Figure 4B) and isoflurane (inset in Figure 4C) anesthesia. Comparing the median (per mouse) effect sizes revealed a significant difference between the three types of anesthesia (Figure 4D). All regimens reduced the basal ratios more strongly in mABCs than in ResGABA cells, but a significant difference was seen only for K/X. Also, at the single-cell level all regimens significantly reduced the basal Twitch-2B ratios in mABCs and ResGABA cells (Figure S5). Under K/X anesthesia, the reduction in mABCs was significantly stronger (Figure S5H).

Figure 4.

Figure 4

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Basal Twitch-2B Ratios of mABCs and ResGABA Cells in Anesthetized Mice

(A–C) Cumulative probability histograms showing the distributions of Twitch-2B ratios under MMF (A), K/X (B), and isoflurane anesthesia (C). The distributions were similar under MMF (A) (p = 0.24, n = 101 versus 217 cells, here and below Kolmogorov-Smirnov test) or isoflurane (C) (p = 0.99, n = 106 versus 232 cells) anesthesia but differed significantly under K/X anesthesia (B) (∗p = 1.2 × 10−3, n = 79 versus 221 cells). Pie charts show the fractions of spiking, non-spiking, and uncertain cells. The fractions were similar under MMF (A) but differed significantly under K/X (B) and isoflurane (C) anesthesia (p = 0.12, 3 × 10−3 and 1.6 × 10−2, respectively, chi-square test).

(D) Boxplot shows the median (per mouse) effect sizes for mABCs and ResGABA cells under three anesthesia regimens (∗p < 0.01, two-way ANOVA with a Bonferroni post-hoc test).

Odor-Response Properties of mABCs and ResGABA Cells under Anesthesia

Next, we compared the odor-evoked responses of mABCs and ResGABA cells under MMF anesthesia, under which basal Twitch-2B ratios in both cell types were similar (Figure 4A), thus assuring comparable recording conditions. Cell responses were first measured in awake state and then re-examined under MMF anesthesia (Figure 5). Similar to data obtained in awake mice (Figure 3), the fractions of reliably responding, non-reliably responding, and non-responding cells differed significantly between mABCs and ResGABA cells (Figure 5C). Going from awake to anesthesia, the fraction of reliably responding mABCs increased, while that of ResGABA cells decreased but the observed differences did not reach the level of statistical significance (p = 0.11 and 0.12, n = 83 mABCs and n = 222 ResGABA cells, McNemar-Bowker test).

Figure 5.

Figure 5

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Odor-Evoked Responses of mABCs and ResGABA Cells under MMF Anesthesia

(A and B) Sample Ca2+ transients evoked by ETI (1.7% of saturated vapor) in an mABC (A) and a ResGABA cell (B).

(C) Pie charts showing the fractions of reliably responding, non-reliably responding, and non-responding cells among mABCs (top) and ResGABA cells (bottom; p = 10−4, chi-square test; n = 83 mABCs, 7 mice and 222 ResGABA cells, 5 mice).

(D–F) Cumulative probability histograms showing the distributions of the CV (D) (p = 0.31, here and below Kolmogorov-Smirnov test, n = 44 and 159 reliably responding cells), the amplitude (E) (p = 0.46), and the maximal odor-evoked Twitch-2B ratios (F) (∗p = 8.7 × 10−5).

(G) Relationships between the basal and the maximal odor-evoked Twitch-2B ratios (mABCs: r = 0.29, p = 5.1 × 10−2; ResGABA cells: r = 0.41, p = 10−7, Spearman's rank correlation).

The CVs in mABCs and ResGABA cells were similar under MMF anesthesia (Figure 5D). The response amplitudes also showed the overall similarity (Figure 5E), but comparison of cells with ΔR/R amplitudes above 200% revealed a significant difference (p = 0.04, Kolmogorov-Smirnov test, n = 15 mABCs and n = 41 ResGABA cells). Consistently, the odor-evoked maximal ratios were significantly higher in mABCs (Figure 5F). While ResGABA cells maintained a linear relationship between the basal and the corresponding maximal ratios (Figure 5G), mABCs lost this relationship under MMF anesthesia.

Differential Effects of Cholinergic and Serotonergic Receptor Blockers on Basal Ca2+ Levels of mABCs and ResGABA Cells in Awake Mice

Because activity of both mABCs and ResGABA cells was significantly reduced under anesthesia, we tested whether centrifugal projections arising from ARAS (ascending reticular activating system) centers (e.g., locus coeruleus, nucleus basalis of Meynert, raphe nuclei) contribute to the neuronal activity observed in awake state and whether these projections target mABCs and ResGABA cells differently. Hence, respective basal Twitch-2B ratios were measured before and during topical applications of noradrenergic (NA), serotonergic (5-hydroxytryptamin [5-HT]), or cholinergic (acetylcholine [ACh]) receptor blockers to the dorsal surface of the OB.

As all blockers were diluted in the HEPES-buffered Ringer's solution, we first assured that this solution does not change the Twitch-2B ratios (Note S1). The basal ratios of mABCs did not change in the presence of the adrenergic receptor blocker prazosin (Figure 6A), whereas the 5-HT receptor blocker methysergide significantly reduced the basal ratios, decreasing the fraction of spiking and increasing the fraction of non-spiking cells (Figure 6B). Similarly, a mixture of scopolamine/mecamylamine significantly reduced the basal ratios and thus the fraction of spiking cells (Figure 6C). The median (per mouse) effect size for prazosin was 2.28%, for methysergide 47.19%, and for mecamylamine/scopolamine 50.40% (p = 0.40, 0.03, and 0.03, respectively, one-tailed Wilcoxon signed rank test, n = 5 mice; Figure 6D). We did not observe any linear correlation between the effect size of the methysergide-evoked blockade in individual mABCs and the basal Twitch-2B ratios of these cells measured under control conditions (Pearson correlation coefficient r = 0.08, p = 0.69). This suggests the absence of a larger 5-HT input to highly spontaneously active mABCs.

Figure 6.

Figure 6

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Effect of Receptor Blockers on Basal Twitch-2B Ratios of mABCs in Awake Mice

(A–C) Cumulative probability histograms showing the distributions of basal Twitch-2B ratios in control and during topical application of prazosin (A), methysergide (B), and mecamylamine/scopolamine (C). Pie charts (A–C, insets) show the fractions of spiking, non-spiking, and uncertain cells under two conditions. Prazosin had no effect (A) (p = 0.37; here and below McNemar-Bowker test, n = 35 cells, 5 mice) but methysergide (B) (p = 3 × 10−3) and mecamylamine/scopolamine (C) (p = 2 × 10−3) changed the fractions significantly.

(D) Boxplot showing the median (per mouse) effect sizes for different receptor blockers (5-HT versus ACh receptor blockers: p = 0.81, Wilcoxon signed rank test).

In ResGABA cells, neither prazosin (Figure 7A) nor methysergide (Figure 7B) reduced the basal Twitch-2B ratios but the ratios were significantly reduced by the ACh receptor blockers (Figure 7C). The median effect size (Figure 7D) for prazosin was 0.53%, for methysergide 6.75%, and for mecamylamine/scopolamine 54.26% (p = 0.5, 0.06, and 0.03, respectively, one-tailed Wilcoxon signed rank test, n = 5 mice). Thus, the 5-HT receptor blocker affected mABCs and ResGABA cells differently (Figure 7E), whereas the effect sizes of ACh receptor blockers were similar.

Figure 7.

Figure 7

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Effect of Receptor Blockers on Basal Twitch-2B Ratios of ResGABA Cells in Awake Mice

(A–C) Cumulative probability histograms showing the distributions of the basal Twitch-2B ratios in control and during topical application of prazosin (A), methysergide (B), and mecamylamine/scopolamine (C). Pie charts (A–C, insets) show the fractions of spiking, non-spiking, and uncertain cells. Prazosin (A) (p = 0.51, here and below McNemar-Bowker test, n = 176 cells, 5 mice) and methysergide (B) (p = 0.11) had no effect, but mecamylamine/scopolamine changed the fractions significantly (C) (p < 10−3).

(D) Boxplot shows the median (per mouse) effect sizes for different receptor blockers.

(E) Comparison of median (per mouse) effect sizes between mABCs and ResGABA cells. There was a significant difference for methysergide (∗p < 10−3, two-way ANOVA with a Bonferroni post-hoc test).

Discussion

This study compared the in vivo functional properties of mABCs with that of ResGABA cells in awake mice. Several unique functional properties of mABCs distinguished them from ResGABA cells. First, basal Twitch-2B ratios, reflecting the basal levels of [Ca2+]i, were significantly higher in mABCs (Figure 2C), with a unique cluster of highly spontaneously active mABCs (Figure 2E), comprising approximately a quarter of the population. Cells with such high levels of activity were absent among ResGABA cells. Secondly, a significantly smaller fraction of mABCs responded to odorants under awake conditions and 2.2 times as many mABCs as ResGABA cells were non-reliably responding (Figure 3C). Because the reliability of mABC odor responses increased under subsequent anesthesia, we hypothesize that some waking-specific inhibitory inputs fall away under anesthesia. Consistently, in reliably responding mABCs of awake mice the response amplitudes were significantly smaller compared with those of reliably responding ResGABA cells. Thirdly, mABCs were in general more sensitive to anesthesia, with significantly lower basal Twitch-2B ratios recorded under K/X anesthesia (Figure 4). Finally, in the awake state the ongoing activity of mABCs but not ResGABA cells was strongly driven by 5-HT inputs, as a 5-HT receptor blocker dramatically reduced basal Twitch-2B ratios only in mABCs.

Methodological Considerations

Correct functional comparison of mABCs and ResGABA cells requires that (1) cell populations are compared a long time after they were labeled with a functional marker and (2) the composition of the populations under study is comparable. To fulfill these criteria we used Twitch-2B, which is well tolerated by living cells and does preserve its functional properties in vivo for months (Thestrup et al., 2014). As ABCs mature to become GABAergic interneurons, we compared them with resident Viaat-positive cells, more than 98% of which are GABAergic interneurons (Chao et al., 2010). It has to be noted that neither mABC nor ResGABA cell populations are homogeneous. According to our immunohistochemical data (Figure S2), ResGABA cells studied here comprise 23.6% of tyrosine hydroxylase-, 38.5% of calretinin-, and 19.8% of calbindin-D-28k-positive cells. These results closely match the literature data reporting 16%–21% of tyrosine hydroxylase-, 33%–44% of calretinin-, and 10%–14% of calbindin-D-28k-positive cells among ResGABA cells (Kosaka and Kosaka, 2007; Panzanelli et al., 2007; Parrish-Aungst et al., 2007). According to our previous data, mABCs comprise 17.4% of tyrosine hydroxylase-, 37.3% of calretinin-, and 16.2% of calbindin-D-28k-positive cells (Kovalchuk et al., 2015). Despite the above similarity, some differences between mABCs and ResGABA cells still exist. For instance, 2.5% of the dopaminergic ResGABA cells are generated only prenatally and are therefore absent among mABCs (Galliano et al., 2018). Instead, mABCs contain ~5% of glutamatergic neurons (Brill et al., 2009), which are absent among ResGABA cells. Therefore, a small mismatch in the composition of the cell populations is possible but, in our view, it cannot explain the population-wide functional differences observed in this study.

Furthermore, we used lentiviruses to label mABCs. Although providing superior cell brightness, this is “a somewhat ‘noisy’ method with regard to precise birth dating” (Livneh and Mizrahi, 2011). Indeed, 5.5% of mABCs in our study were doublecortin-positive and thus likely younger than their nominal age. However, the known properties of young ABCs are opposite to the herein described properties of mABCs. Whereas mABCs show higher basal Twitch-2B ratios compared with resident cells, reflecting their high spontaneous firing rate (Figures 1, 2, and S1), the spontaneous firing rate of ABCs at 9–28 days post-injection is much lower than that of resident cells (Kovalchuk et al., 2015; Livneh et al., 2014). Secondly, immature ABCs have higher odor responsiveness compared with resident cells (see Figures 3B and 3D in Livneh et al., 2014), whereas mABCs have significantly lower odor responsiveness (Figure 3C). Finally, the amplitudes of odor-evoked signals of immature ABCs are higher compared with resident cells (see Figure 3C in Livneh et al., 2014), whereas in our study, the amplitudes of >60% of mABCs are significantly lower (Figure 3E). These data strongly suggest that the use of lentivirus-based cell labeling, if anything, underestimates the differences between mABCs and ResGABA reported in the present study.

Ongoing Activity of GABAergic Juxtaglomerular Neurons

The use of the ratiometric Ca2+ indicator allowed to visualize the ongoing spontaneous activity of JGCs. Only 18%–25% of JGCs were non-spiking, whereas the vast majority of both resident and adult-born JGCs were active in awake state. JGCs thus impose a strong ongoing inhibitory drive on their synaptic partners. Interestingly, 24.1% of mABCs had very high basal Twitch-2B ratios, likely caused by ongoing spontaneous activity and AP firing (Figure 1D). Such a subpopulation was absent among ResGABA cells. Previously, the ongoing spontaneous activity was reported for “many” OB GCs in awake mice (Kato et al., 2012) but was not described by the same authors for the population of parvalbumin-positive interneurons located in the external plexiform layer (Kato et al., 2013). However, the use of a non-ratiometric Ca2+ indicator in previous studies allowed to visualize fluctuations of ongoing activity rather than its steady-state level (see also Figure 3 in Homma et al., 2013).

All three anesthesia regimens used in our study significantly reduced the ongoing spontaneous activity in JGCs, suggesting that this effect is caused by the change in brain state. Under anesthesia, the fraction of silent ResGABA cells roughly doubled, while that of silent mABCs increased by ~3.5-fold. However, approximately 25% of ResGABA cells remained active, in contrast to OB GCs, showing little spontaneous activity both under urethane/chlorprothixene and K/X anesthesia (Kato et al., 2012). Consistent with our data, low-resolution imaging of population activity in GAD2-Cre and TH-Cre mice, in which either PGCs or SACs are selectively labeled, revealed an isoflurane-induced decrease in resting GCaMP3 fluorescence by approximately 20% (Wachowiak et al., 2013).

For ketamine/xylazine, the extent of the anesthetic-mediated blockade was significantly stronger for mABCs compared with ResGABA cells (Figures 4D and S5). This effect is likely caused by the NMDA receptor blocker ketamine, because xylazine acts alike the MMF anesthesia component medetomidine and MMF anesthesia had a similar effect on mABCs and ResGABA cells. In ABCs, NMDA receptors are important for differentiation, synaptic development, maturation of glutamatergic synapses, and long-term cell survival (Kelsch et al., 2012; Lin et al., 2010; Platel and Kelsch, 2013; Tashiro et al., 2006). NMDA receptors are thought to underlie the heightened synaptic plasticity of ABCs, which is restricted to the critical period of ABC development (age 4–8 weeks; Ge et al., 2007; Tashiro et al., 2006). Our data suggest that also after this period ABCs preserve the heightened levels of NMDA receptor activity.

Odor-Evoked Responsiveness of GABAergic Juxtaglomerular Cells

In contrast to the robust spontaneous activity of mABCs, their ETI-evoked responses in the awake state involved fewer cells, and were weaker and much less reliable than those of ResGABA cells. However, the response reliability in the very same mABCs improved under the anesthesia. Thus, the lack of reliability in the awake state cannot be explained by the fewer connections to the synaptic partners, i.e., weaker integration of these cells into the surrounding network. Rather, it is either due to a specific inhibitory input active only in the awake state or to a specific excitatory input active under the anesthesia. The majority of PGCs and SACs receive their olfactory input indirectly, via glutamatergic external tufted (Kiyokage et al., 2010) or M/T cells (Lledo et al., 2008). Furthermore, OB glutamatergic cells seem to be less sensitive to anesthesia compared with GABAergic cells (Kato et al., 2012; Rinberg et al., 2006; Wachowiak et al., 2013). Thus, an increase in the reliability of odor-evoked responses in mABCs could be explained by the anesthesia-induced relative increase in the excitation/inhibition ratio. In any case, in the awake state mABCs are unlikely to play the major role for the reliable feedforward inhibition of odor-evoked responses in M/T cells as they are responding less. According to our data, this role is much better fulfilled by ResGABA cells.

Neuromodulatory Inputs to Juxtaglomerular Neurons

This is the first study analyzing the influence of ARAS-related neuromodulators on the ongoing activity of the adult-born and resident JGCs in the awake state. In line with anatomical data showing that noradrenergic projections from the locus coeruleus terminate in all but the most superficial layers of the OB (Devore and Linster, 2012; Gomez et al., 2005), the adrenergic receptor blocker had little effect on the ongoing activity of JGCs (Figures 6 and 7). We have, however, observed a strong effect of the ACh receptor blockers. Also these data are consistent with the anatomy of the cholinergic projections from the basal forebrain, known to innervate primarily glomerular and GC layers (Devore and Linster, 2012; Gomez et al., 2005). While in single unit recordings in anesthetized rats (Ravel et al., 1990) iontophoretic application of ACh elicited both excitatory and inhibitory responses, in our hands the effect of endogenous ACh was clearly excitatory as its blockade roughly doubled the fraction of non-spiking JGCs. However, the effects of ACh blockers, and hence also the density of functional cholinergic projections synapsing on mABCs and ResGABA cells, were similar, suggesting that in the awake state both cell types are working on the same common project. Experiments in anesthetized rats (Chaudhury et al., 2009) as well as computational analyses (Devore and Linster, 2012) suggested that endogenous cholinergic activation of these cells might contribute to amplitude-invariant processing of odor-evoked signals, decorrelation of odor representations and an increase in perceptual discrimination between chemically similar odorants.

The Unique Serotonergic Innervation of mABCs

The 5-HT inputs to the OB arise from the dorsal raphe nucleus and predominantly target the GL (Gomez et al., 2005; McLean and Shipley, 1987). They inhibit inputs from the nose, partly via JGC-mediated presynaptic inhibition (Petzold et al., 2009; Wachowiak et al., 2009). Consistently, in awake mice the ongoing spontaneous activity of mABCs was blocked by methysergide (Figure 6B), suggesting that 5-HT inputs do sustain the ongoing inhibition that these cells impose on their synaptic partners. Strikingly, the sensitivity to methysergide, one of the most potent modulators of the presynaptic odor-evoked responses (Petzold et al., 2009), represented a unique feature of mABCs and was barely seen in ResGABA cells (Figure 7E), suggesting that this important function is fulfilled by adult-born JGCs. Because the activity of 5-HT neurons is brain state dependent with high activity levels during awake resting but not during attentive states of sensory acquisition (Jacobs and Azmitia, 1992; Jones, 2005), the mABC-mediated presynaptic inhibition is likely to be brain state specific, actively inhibiting the OB inputs during rest and releasing the inhibition during focused attention. These data support the hypothesis that ABCs function as coincidence detectors between the behavioral state of the animal (e.g., arousal, attention, expectation) and sensory inputs arising from the environment (Lazarini and Lledo, 2011).

Interestingly, 5-HT modulates the fate and the functional state of ABCs throughout the entire life. It promotes proliferation of progenitor cells in the SVZ (Banasr et al., 2004; Brezun and Daszuta, 1999), controls the velocity and the directionality of ABC migration in the RMS (Garcia-Gonzalez et al., 2017), and likely influences the survival of these cells in the OB (Soumier et al., 2010). Here, we show that ABCs do preserve their privileged 5-HT inputs even after integration into the OB circuitry and maturation therein.

In conclusion, our study is the first to describe the in vivo functional properties of mature adult-born JGCs in awake mice, showing that even under control conditions these properties are different from those of ResGABA cells. Together with data of Livneh et al. (2014), showing that ABCs can acquire distinct properties when developing in the odor-enriched environment, our data firmly identify mABCs as a neuronal population with distinct and maybe even unique functions.

Experimental Procedures

Animals

Experiments were conducted in accordance with biometrical planning/institutional animal welfare guidelines and were approved by the state government of Baden-Württemberg, Germany. C57BL/6 (WT, Charles River) and B6.FVB-Tg(Slc32a1-cre)2.1Hzo/FrkJ (Viaat-Cre bred on the C57BL/6 background, Jackson Laboratory) mice of either sex were used to study mABCs and ResGABA cells, respectively. Animals were kept in pathogen-free conditions at 22°C, 60% air humidity, 12-h light/dark cycle, with ad libitum access to food and water. Females stayed in groups of three to five mice, males were kept individually.

Expression of Twitch-2B in mABCs and ResGABA Cells

To label ABCs, house-made lentiviral (Note S2) vectors (1.5 μL, 1 × 108 infectious units/mL; Maslyukov et al., 2018) encoding the Ca2+ indicator Twitch-2B under the ubiquitin promoter were stereotactically injected (3.0 mm anterior, 0.84 mm lateral from bregma, and 2.9 mm ventral from dura) into the RMS of adult WT mice. By the time studied, only 5.5% ± 6.7% (median ± SEM, n = 448 cells, 5 mice) of Twitch-2B-expressing mABCs also expressed doublecortin, a marker for immature neurons.

To label ResGABA cells, adeno-associated virus (1 × 1013 infectious units/mL, 1 μL of 1:7 diluted solution) encoding a Cre-inducible version of Twitch-2B under control of the CAG promotor (AAV1.CAG.Flex.Twitch2B.WPRE.SV40, Addgene 49531M; Penn Vector Core, Philadelphia, PA) was injected into the dorsal OB (at 0.35, 0.25, and 0.15 mm depth, ~300 nL per injection) of 1-month-old Viaat-Cre mice at an 45° angle from the horizontal plane.

In Vivo Two-Photon Ca2+ Imaging

Mature ABCs (3.5 ± 1.6 months post-injection) and ResGABA cells (8.9 ± 1.6 months post-injection) were imaged in awake/anesthetized animals (aged 7.6 ± 1.9 and 9.8 ± 1.6 months, respectively). Before awake imaging, mice were habituated to the setup for ~12 days by daily fixations, lasting 5 min (at the beginning) to 60 min (at the end of training).

For anesthesia induction, mice were sedated with isoflurane (1–2 min, 2.5% in O2) and then injected intraperitoneally with either K/X or MMF. The breathing rate was monitored with a pressure sensor (ADInstruments, Spechbach, Germany) attached to the back. For isoflurane anesthesia, it was kept at 100–130 breaths/min by adjusting the isoflurane concentration (0.9%–1.5% in O2). Recordings commenced 10 min after anesthesia induction.

We used an Olympus FluoView 1000 laser scanning system coupled to a mode-locked Ti:Sapphire laser (Mai Tai DeepSee, Spectra-Physics, Germany). Twitch-2B was excited at 890 nm and imaged using a 20× UMPlan FI 1.0 NA water-immersion objective (Zeiss). Fluorescence of mCerulean3 and cpVenusCD was separated using a 515-nm dichroic mirror and a 475/64-nm band pass and 500-nm-long pass filters, respectively. Time series were recorded at 4–8 Hz. In some experiments, basal Twitch-2B ratios were derived from single frames of Z series. Z series were acquired at a depth of 10–120 μm: image size, 640 × 640 pixels; Kalman filter, 2; and step size, 2 μm.

ETI (Sigma-Aldrich) was applied to the mouse snout using a custom-built flow dilution olfactometer at a flow rate of 300 mL/min (Homma et al., 2013). The odorant was delivered eight times for 4 s per trial with an inter-trial interval of 1–2 min.

Application of Receptor Blockers in Awake Mice

Blockers were dissolved in HEPES-buffered Ringer's solution (Note S1). Prazosin, methysergide, scopolamine, and mecamylamine were applied at final concentrations of 100 μM, and 4, 50, and 115 mM, respectively.

Data Analyses

The regions of interest (ROIs) covering cell somata were drawn manually in ImageJ 8 (https://imagej.nih.gov/ij), the background ROI had a comparable size. Further analyses used Custom written MATLAB scripts (The MathWorks). The ratio was calculated as

Ratio=mCerulean3−mCerulean3backgroundcpVenusCD−cpVenusCDbackground (Equation 1)

using the mean ROI intensities measured in the respective channels.

Basal ratios are averages of all ratio values measured in a single time-series recording in the absence of any exogenous stimuli. Time points of recording were chosen randomly, no selection based on cell’s instant activity pattern or any other criteria was performed.

Maximal ratios (Rmax) of the odor-evoked Ca2+ transients were measured 0–6 s after odor stimulus onset, after smoothing the ratio trace twice with the 0.3-s time window binomial filter.

ΔR/R was calculated as follows:

ΔRR(%)=Ratio−RatiobaselineRatiobaseline∗100,

where Ratio is the trace calculated according to (1) and Ratiobaseline is a mean ratio measured 1–5 s before the onset of the odor stimulus.

Odor-evoked signals were scored as responses when the signal amplitude was at least 15% of ΔR/R and six times larger than the SD of the corresponding baseline noise. The CV was calculated as follows:

CV=SDx¯∗(1+14∗n),

where SD is the standard deviation of Rmax, x¯ is the mean Rmax of the successful trials, and n is the number of successful trials. The part in parentheses is the correction for the small sample size.

The effect size was calculated as:

effectsize%=Rctr−RtestRctr−Rmin  x 100,

where Rctr and Rtest are the cell's ratios measured under control and test conditions, respectively, Rmin = 1.25 is the lowest ratio ever observed in our recordings. Rtest = Rmin was considered as a 100% block of cell activity. As activity can be blocked in spiking cells only, the effect size was calculated only for cells with ratios >2.4.

Statistical Analyses

Two-sided statistical tests were done using MATLAB, GraphPad Prism (www.graphpad.com), SPSS, or Vassar Stats (http://vassarstats.net/). Normality of the data distribution was tested with a one-sample Kolmogorov-Smirnov test. p values < 0.05 were considered significant. Unless indicated, data are presented as median ± IQR.

Data and Code Availability

All data of this study are reported in the main text and Supplemental Information. In-house MATLAB scripts and the raw imaging files are available from the corresponding author upon reasonable request.

Author Contributions

O.G. conceived the study. N.F.-T., Y.K., S.F., N.M., E.Z., and A.A. performed the experiments and/or data analyses. N.F.-T. and O.G. wrote the manuscript and all authors approved the manuscript.

Acknowledgments

We thank A. Weible and K. Schöntag for technical assistance; Y. Liang and W. Pfleging (Institute for Applied Materials, Karlsruhe, Germany) for help with topical drug application; X. Su, K. Li, N. Asavapanumas, and M. E. Schmidt for help with experiments/data analyses. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grant no. GA 654/14-1 (to O.G.).

Published: November 19, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.stemcr.2020.10.010.

Supplemental Information

Document S1. Supplemental Experimental Procedures, Note S1 and S2, Figures S1–S5, and Table S1
mmc1.pdf (766KB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (2.6MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Experimental Procedures, Note S1 and S2, Figures S1–S5, and Table S1
mmc1.pdf (766KB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (2.6MB, pdf)

Data Availability Statement

All data of this study are reported in the main text and Supplemental Information. In-house MATLAB scripts and the raw imaging files are available from the corresponding author upon reasonable request.

Articles from Stem Cell Reports are provided here courtesy of Elsevier

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